Stem cell-based cell replacement therapy is a potentially novel method to treat Parkinson's disease (PD). Our recent progress reveals molecular and cellular mechanisms underlying development and specification of midbrain dopaminergic (DA) neurons by genetic modification of embryonic stem (ES) cells using key transcription factors, such as Nurr1 and Pitx3. Nurr1 expression increases the number of all ES cell-derived DA midbrain-like neurons. In contrast, Pitx3 does not affect the total number of TH+ neurons, but increases the proportion of A9-Iike DA neurons, indicating that Nurr1 and Pitx3 may have co-operative, but distinct roles in midbrain DA neuron development In this proposal, based on the above progress, we propose to generate transgenic ES cell lines that express both Nurr1 and Pitx3. By comparing these new ES cells with empty vector-transduced, Nurr1-expressing, and Pitx3-expressing ES cell lines, we will test whether these genetic modifications can enhance differentiation to the A9-like DA neuronal fate in vitro and in vivo. In addition, we will also pay attention to how epigenetic control may regulate the differentiation of ES cells and its stability. Critically, we will also purify (by FACS in Prof. Isacson's laboratory) ES-derived neural progenitor cells or immature post-mitotic DA neurons using the Sox1-green fluorescent protein (GFP) knock-in ES cell line, a DA-specific promoter fused to the GFP gene, and TH-GFP knock-in ES cell Ones. To translate this work into a realistic therapeutic modality for PD, we use human ES cells similarly modified by genetic manipulation to facilitate their differentiation and/or maturation into the midbrain A9 fate and experiment with them using both in vitro differentiation assays and in vivo transplantation surgery in rodents. The investigators in Project 6, using their non-human primate model of PD will test these human DA neurons, derived from our ES cell work. In all of the PD focused in vivo experiments, the human ES derived post-mitotic DA neurons will be analyzed using a battery of behavioral paradigms, functional assays (also assisted by UCLA NIH PD Udall Center) and convergence with post-mortem stereological data (assisted by our neurohistology core, for human markers and stereology). In summary, this Project in the Harvard-Mclean PD Udall Center provides a platform of genetic technology and provides insights into critical issues for using genetically modified ES cells for transplantation therapy of PD. We will focus on differentiation and specification of ES cells to the desirable midbrain A9-like DA neurons and their effects in PD models. Using human ES cells derived DA neurons, the proposed experiments of Aim 2 will eventually translate into a cell-based therapy of PD.